1. Field of the Invention
The present invention relates to a method for secretory production of a heterologous protein.
2. Brief Description of the Related Art
Secretory production of heterologous proteins by microorganisms has been previously reported, and includes secretory production by a Bacillus bacterium (Microbiol. Rev., 57, 109-137 (1993)), methanol-assimilating yeast, Pichia pastoris (Biotechnol., 11, 905-910 (1993)), filamentous fungi of the genus Aspergillus (Biotechnol., 6, 1419-1422 (1988) and Biotechnol., 9, 976-981 (1991)), and so forth.
Coryneform bacteria has also been used for the secretory productions of heterologous proteins, and examples include the secretion of a nuclease and a lipase by Corynebacterium glutamicum (henceforth also abbreviated as C. glutamicum) (U.S. Pat. No. 4,965,197, J. Bacteriol., 174, 1854-1861 (1992)), secretion of a protease such as subtilisin (Appl. Environ. Microbiol., 61, 1610-1613 (1995)), secretion of a protein using signal peptides of cell surface layer proteins PS1 and PS2 (also referred to as CspB) of coryneform bacteria (Japanese Patent Laid-open (Kohyo) No. 6-502548), secretion of a fibronectin-binding protein using the signal peptide of PS2 (CspB) (Appl. Environ. Microbiol., 63, 4392-4400 (1997)), secretion of protransglutaminase using signal peptides of PS2 (CspB) and SlpA (also referred to as CspA) (Japanese Patent No. 4320769), secretion of a protein using a variant type secretion system (Japanese Patent Laid-open (Kokai) No. 11-169182), secretion of a protransglutaminase by a variant strain (Japanese Patent No. 4362651), secretion of a protein using a Tat-dependent signal peptide (Japanese Patent No. 4730302), and so forth.
It has also been reported that when producing a heterologous protein connected to a signal peptide, a sequence of one or more amino acid residues from the N-terminal sequence of a cell wall protein of a Bacillus bacterium should be inserted between the signal peptide and the heterologous protein (Japanese Patent Laid-open (Kokai) No. 11-341991 and Japanese Patent Laid-open (Kokai) No. 2000-316579).
CspB (PS2) is a cell surface layer protein found in C. glutamicum (Mol. Microbiol., 9, 97-109 (1993)). There are Corynebacterium bacterium strains having a homologue of the cell surface layer protein CspB, and those that do not have such a CspB homologue. Amino acid sequences of CspB homologues have been reported for 28 strains of C. glutamicum (J. Biotechnol., 112, 177-193 (2004)). When comparing the N-terminal amino acid sequences of the CspB homologues from these 28 strains, it was found that both the signal sequences of 30 amino acid residues, as well as the N-terminal 3 amino acid residues (Gln-Glu-Thr) of the mature proteins, were completely conserved (J. Biotechnol., 112, 177-193 (2004)).
As described above, methods for secretory production of a heterologous protein connected to the signal peptide of CspB (PS2) are known (for example, Japanese Patent Laid-open (Kohyo) No. 6-502548 and Japanese Patent No. 4320769, Appl. Environ. Microbiol., 63, 4392-4400 (1997)). Moreover, it has also been reported that, by inserting 1, 14, or 38 amino acid residues of the N-terminus of the mature CspB protein of C. glutamicum ATCC 13869 (SEQ ID NO: 96) between the signal peptide and the heterologous protein (protransglutaminase), the heterologous protein can be produced by secretory production, with the 38 amino acid residues insertion providing an increased secretory production amount of the protransglutaminase (Japanese Patent No. 4320769). However, inserting an amino acid sequence that includes Gln-Glu-Thr between the signal peptide and the heterologous protein for expression of the heterologous protein has not been previously reported.
It has been reported that the N-terminal amino acid residue of the mature cell surface layer protein CspB of C. glutamicum is blocked by the Edman degradation, and thus it has been suggested that the original N-terminal amino acid residue of glutamine is converted into a pyroglutamic acid residue (Mol. Microbiol., 9, 97-109 (1993)). However, it has not been previously reported that when expressing a heterologous protein in which an amino acid sequence including Gln-Glu-Thr is inserted between a signal peptide and the heterologous protein, the N-terminal glutamine residue of the heterologous protein can be converted into a pyroglutamic acid residue.